Detection of biological molecules using chemical amplification and optical sensors

ABSTRACT

Methods are provided for the determination of the concentration of biological levels of polyhydroxylated compounds, particularly glucose. The methods utilize an amplification system that is an analyte transducer immobilized in a polymeric matrix, where the system is implantable and biocompatible. Upon interrogation by an optical system, the amplification system produces a signal capable of detection external to the skin of the patient. Quantitation of the analyte of interest is achieved by measurement of the emitted signal. Specifically, the analyte transducer immobilized in a polymeric matrix can be a boronic acid moiety.

This application is a Continuation Application of and claims the benefitof U.S. patent application Ser. No. 08/752,945, filed Nov. 21, 1996, nowU.S. Pat. No. 6,011,984, which is a Continuation-In-Part of U.S.Provisional Application Ser. No. 60/007,515, filed Nov. 22, 1995, and isrelated to U.S. patent application Ser. No. 08/721,262, filed Sep. 26,1996, now U.S. Pat. No. 5,777,060 which is a Continuation-In-Part ofU.S. patent application Ser. No. 08/410,775, filed Mar. 27, 1995, nowabandoned the disclosures of which are incorporated by reference.

The United States Government may have rights in inventions disclosed inthis application pursuant to Contract No. W-7405-ENG-48 between theUnited States Department of Energy and the University of California forthe operation of Lawrence Livermore National Laboratory.

FIELD OF THE INVENTION

This invention relates generally to biological sensors. Morespecifically, this invention relates to minimally invasive amplificationsystems and optical sensors capable of detecting polyhydroxylatedcompounds such as glucose.

BACKGROUND OF THE INVENTION

An essential tool for the care of the diabetic patient is themeasurement of blood glucose. Recently, the NIDDK (National Institutesof Diabetes and Digestion and Kidney Diseases) has released the resultsof a large clinical trial, the DCCT (Diabetes Control and ComplicationsTrial) that shows conclusively that improved blood glucose controlreduces the risk of long term complications of diabetes. See, DCCTResearch Group, N. Engl. J. Med. 329:977-986 (1993).

Current technology requires that a blood sample be obtained formeasurement of blood glucose levels. Samples of venous blood can beobtained from the patient for this measurement, but this method islimited to only a few samples per day, and is not practical for the careof outpatients.

Self monitoring of capillary blood glucose is practical, but stillrequires multiple and frequent skin punctures. Consequently, mostpatients perform 2-6 tests per day depending on their personalcircumstances and medical condition. Self monitoring results areinfluenced by technique errors, variability of sample volume andimpaired motor skills (important with hypoglycemic episodes). Thepatient must interrupt other activities to perform the task of bloodglucose measurement.

The concept of an implantable sensor to continuously measure the glucoselevels in holter monitor type applications and in ambulatory diabeticindividuals has existed for several decades. For a recent discussion,see Reach, et al., Anal. Chem. 64:381-386 (1992). The primary focus hasbeen to overcome the disadvantages of capillary blood glucose selfmonitoring by developing a glucose sensor, which at the very least,would provide more frequent and easily acquired glucose information. Inaddition, the sensor could function as a hypoglycemic and hyperglycemicalarm, and ultimately serve as the controller for an artificialendocrine pancreas. The potential limitations of this approach includethe limited life of the enzyme, glucose oxidase, the limited lifetime ofthe sensor (2-3 days), and the need to wear the device.

The concept of a non-invasive glucose sensor has received significantmedia and technical attention over the past several years. The basicscientific goal has been to utilize near infrared (NIR)spectrophotometry to detect the absorbance properties of the glucosemolecule. The inherent problem with this approach is that the glucosesignal is weak and is masked by other body constituents. Moreover, if itis possible to detect glucose, the system will most likely rely uponexpensive optics and significant computing power, resulting in a large,expensive device which requires frequent recalibration to the patientand provides intermittent data.

Some of the approaches to non-invasive blood glucose measurement aredescribed in U.S. Pat. Nos. 4,428,366, 4,655,225, 4,805,623, 4,875,486,4,882,492, 5,028,787, 5,054,487, 5,070,874, 5,077,476, 5,086,229, and5,112,124, the disclosures of each being incorporated herein byreference.

Most of these approaches involve the use of transdermal infrared or nearinfrared radiation in either a transmission or reflectant mode. In spiteof the large number of patents and intense efforts by at least thirtymajor companies, no devices have been successfully implemented in thefield.

The problems with these approaches are well known and described indetail by Marquardt, et al., Anal. Chem., 65:3271 (1993) and Arnold, etal., Anal. Chem., 62:1457 (1990). Marquardt, et al. have shown that in asimple aqueous solution, the absorbance of a 13 mM glucose solution (234mg/dl) gave a signal with a S/N ratio of about 2. In a proteincontaining matrix, the actual signal from glucose cannot be detectedwithout considerable manipulation of the data using a partial leastsquares approach. Such small signal to noise ratios are not practicalfor developing robust simple instrumentation. Furthermore, the deviceused in this research is a large spectrophotometer that must be able toscan over reasonably broad wavelength ranges.

In contrast to these purely non-invasive optical approaches, an implantcontaining a transducer chemical whose optical properties are stronglymodulated by recognition of the target analyte will result in a largeamplification of the optical signal. It is in this sense that the term“chemical amplification” is used throughout this application. Forinstance, U.S. Pat. No. 4,401,122 describes an implanted enzymaticsensor that measures the H₂O₂ produced when glucose and oxygen react inthe presence of the enzyme glucose oxidase. This approach is limited byprofound biocompatibility concerns, particularly changes in stabilityrelated to glucose diffusion to the sensor and the lifetime of an enzymein an implanted environment. Further concerns using enzymes are createdbecause the large differential between O₂ and glucose concentrations inthe body requires a glucose limiting outer membrane. This membranelimits not only the glucose, but the analytical signal as well.

One approach to solving the problems is described in U.S. Pat. No.5,342,789. In this approach, a fluorescent labeled glycoprotein competeswith glucose for binding to a differently fluorescent labeled lectin.Because there is some resonance energy transfer from one label to theother, the presence of glucose reduces the fluorescence intensity of thesystem. There are two major drawbacks to the system as described in the'789 patent. The first problem is that both labels are photoexcited bythe same source; the background signal is significant. The secondproblem is related to the ability of the system to be implanted into thebody. The resonance energy transfer requires diffusion of glucose to thelectin and diffusion of the labeled glycoprotein away from the lectin.In order for the system to have a reasonable time constant forphysiological applications, the reagents must be in solution and free todiffuse via a concentration gradient. This makes the device difficult toimplement reliably since a reservoir must be designed which allowsglucose to diffuse in but prevents the proteins and lectin fromdiffusing out.

Accordingly, there has been a need for a glucose sensor able to measureglucose over the entire physiological range of 30 to 500+ mg/dl (1.6 to28+ mM). It should provide continuous glucose information and be easy touse. The sensor would not require a sample of blood and would be painfree. From an analytical chemistry standpoint, both the accuracy and theprecision would be greater than 95% and the sensor should benon-invasive or minimally invasive. From an instrumental point of view,the device should have a linear dynamic range of at least 200 and asignal to noise ratio of at least 50. Attainment of these figures willensure that analytical precision and accuracy can be achieved. However,less sensitive instruments could be useful providing measurement of theanalyte signals is accurate. The present invention fulfills these needsand provides other related advantages.

SUMMARY OF THE INVENTION

The present invention provides methods for the determination ofbiological levels of polyhydroxylated compounds, particularly glucose.The methods utilize an amplification system which is implantable andwhich produces a signal capable of detection, typically external to theskin of a mammal, for example, a human. The amplification system is ananalyte transducer which is immobilized in a polymeric matrix.Generation of a signal by the amplification system is typically theresult of interrogation by an optical source. Importantly, the signaldoes not require resonance energy transfer, but instead relies onelectron transfer (e.g., molecular electron transfer or photoelectrontransfer). Detection of the signal produced then determines the quantityof polyhydroxylated compound or analyte of interest.

There are therefore two important aspects of the invention. The first isan implantable amplification system (IAS) which includes amplificationcomponents which are immobilized in a polymer matrix, typically abiocompatible matrix, either by entrapment or by covalent attachment.The second aspect of the invention is an optical system whichinterrogates the immobilized amplification components to produce adetectable signal. In some embodiments, the optical system is atransdermal optical system, while in other embodiments a fiber opticsystem is used.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic of the optical glucose monitoring system.

FIG. 2 illustrates a schematic of an optical analyte monitoring systemwhich further illustrates the binding of a polyhydroxylated analyte toan amplification component following permeation into a biocompatiblematrix.

FIG. 3 illustrates one embodiment of the invention which uses a fiberoptic bundle as a “light pipe” for interrogation of an implantedamplification system.

FIG. 4 illustrates another embodiment of the invention which uses asubcutaneous light source for interrogation of an implantedamplification system.

FIG. 5 illustrates another embodiment of the invention which uses asubcutaneous light source and detector to provide a completely subdermalanalyte monitoring system.

FIG. 6 illustrates another embodiment of the invention which uses asubcutaneous light source and detector to provide a completely subdermalanalyte monitoring system which is coupled to an analyte source ormedicament pump (e.g., an insulin pump) to provide a “closed loop”monitoring and supplementation system (e.g. an artificial pancreas).

FIG. 7 shows the chemical reactions of glucose and glucose oxidase toproduce hydrogen peroxide which can be detected optically.

FIG. 8 shows the curves from the reaction shown in FIG. 7, namely thefluorescence intensity as a function of time for a variety of glucoseconcentrations.

FIG. 9 shows the fluorescence spectrum of rhodamine-labeled concanavalinA in different glucose concentrations.

FIG. 10 shows the reversible interaction between a polyhydroxylatedanalyte such as glucose and a boronate complex, N-methyl-N-(9-methyleneanthryl)-2-methylenephenylboronic acid.

FIG. 11 provides the structures for a number of boronate compounds offormula I, along with excitation and emission wavelengths.

FIG. 12 illustrates a synthesis scheme for boronate complexes which areuseful as amplification components.

FIG. 13 illustrates another synthesis scheme for boronate complexeswhich are useful as amplification components.

FIG. 14 provides three examples of implantable amplification systems forthe immobilization of amplification components.

FIG. 15 provides a calibration curve for the quenching of fluorescenceintensity by glucose at pH 7.4.

FIG. 16 shows reversible fluorescence versus glucose concentration foran anthracene boronate solation.

DETAILED DESCRIPTION OF THE INVENTION

The following abbreviations are used herein: dl, deciliter; DEG,diethylene glycol; DMF, dimethylformamide; IAS, implantableamplification system; PBS, phosphate buffered saline; THF,tetrahydrofuran; DI, deionized; PEG, poly(ethylene)glycol; mv,millivolts; mg, milligrams.

General

The broad concept of the present invention is illustrated in FIG. 1. Ascan be seen, the basic scheme utilizes both a detector and source modulewhich can be external to the skin. The source provides an excitationsignal which interrogates a subcutaneous amplification system. Thesystem then produces an amplified signal which is monitored by theexternal detector.

The amplification system can be implanted into a variety of tissues.Preferably, the system is implanted subcutaneously at a depth of from 1to 2 mm below the surface of the skin. At this depth the system is mosteasily implanted between the dermis layer and the subcutaneous fatlayer. These layers, in mammals are relatively easily separated and anamplification system (e.g., chemical amplification components in abiocompatible polymer) can be inserted into a small pocket created in aminor surgical procedure. The implanted system can be profused bycapillary blood and made of a material through which glucose can easilydiffuse. Alternatively, the amplification system can be placed incontact with other fluids containing the analyte of interest.

In one group of embodiments (illustrated in FIG. 1), the amplificationsystem contains an immobilized chemical amplification component whichmay contain a fluorescent moiety providing a signal which is modulatedby the local analyte concentration. A filter can also be incorporatedinto the system for the fluorescent photons (for those embodiments inwhich a fluorescent dye is used). The implanted amplification system isinterrogated transdermally by a small instrument worn or placed over theimplant. The small instrument contains a light source (e.g., a filteredLED) and a filtered detector (e.g., a photomultiplier tube, an unbiasedsilicon photodiode). The signal from the detector provides a continuousreading of the patient's analyte level which can also be used as inputto, for example, an insulin pump or a visual reading for the patient.Alternative embodiments are described below (e.g., use of a fiber opticfor interrogation of the amplification system).

FIG. 2 provides yet another schematic which illustrates theamplification system. According to this figure, the amplification systemincludes a permeable membrane, a matrix for immobilizing theamplification components, and the amplification components themselves.The polyhydroxylated analyte can then permeate the matrix, bind to theamplification components and produce a signal upon interrogation whichis collected, filtered and detected. The optical sources can be avariety of light sources (e.g. laser diode, LED) and the light can bedelivered to the amplification system via delivery methods which couldinclude lenses and fiber optics. Alternatively, the opticalinterrogation can take place with transdermal illumination. Theresultant signal can be collected, again via a fiber optic or lens, andsent to the detector, with the optional use of an intervening filter ordiscriminator.

In addition to the embodiments generally described in FIGS. 1-2, thepresent invention provides sensing systems and methods as generallyillustrated in FIGS. 3-6.

In FIG. 3, a light source is positioned external to the skin and theamplification system is placed at or coated on the distal end of a fiberoptic, which is inserted through the skin into a subcutaneous layer. Thefiber optic serves to conduct the light from the source to theamplification system, and then collects the light emitted from theamplification system and conducts it back to the detector.

Yet another embodiment is provided in FIG. 4. According to this figure,the light source is also implanted under the dermis. Upon interrogationof the IAS by the internal light source, the IAS provides a signal whichis transdermally transmitted to an external detector.

In still another embodiment (FIG. 5), the light source and detector areboth implanted under the dermis. The detector then provides transmissionof the information to an output reading device which is external to theskin.

Finally, for those embodiments in which glucose levels are determined,some aspects of the invention are directed to coupling of the detectorsignal to an insulin pump system in a “closed-loop” artificial pancreas(see FIG. 6).

As a result of the above descriptions, the biosensors of the presentinvention comprise two important components. The first component is animplantable amplification system or IAS, which includes both signalamplification components and a polymer matrix. Additionally, animportant feature of the present invention is the immobilization of theamplification components in the polymer matrix. The immobilization canbe carried out by physical entrapment or by covalent attachment. Thesecond component is the optical system which utilizes transdermal orfiber optic transmission of light or signal.

Implantable Amplification Systems (IAS)

In one aspect, the present invention provides an implantableamplification system which is a combination of an analyte signaltransducer or amplification components and a polymer matrix, preferablya biocompatible matrix. There are several methods for chemicalamplification of an analyte signal, including enzymatic means,equilibrium-binding means and spectroscopic means.

Amplification Components

1. Enzymatic Methods

Enzymatic methods convert glucose stoichiometrically to hydrogenperoxide, which can be affected via fluorescence, chemiluminescence orabsorbance means. One such scheme uses the classical H₂O₂ detectionscheme as described by Guilbault and coworkers. See, Guilbault, et al.,Anal. Chem., 40:190 (1968). In this dimerization based scheme, anoptical signal due to glucose can be amplified and detected optically.The first equation in FIG. 7 shows the reaction of glucose and oxygenwhich is catalyzed by the enzyme glucose oxidase. The products are thelactone which immediately converts to gluconic acid, and hydrogenperoxide (H₂O₂). The second equation in FIG. 7 shows the reaction of thehydrogen peroxide and parahydroxyphenyl acetic acid (HPAA). Thisreaction is catalyzed by another enzyme, horseradish peroxidase (HRP).The product of the reaction is the dimer of the parahydroxyphenyl aceticacid. The dimer is highly fluorescent, and its fluorescence isproportional to the glucose concentration. FIG. 8 shows the curves fromthe reaction shown in FIG. 7. These curves show the fluorescenceintensity as a function of time for a variety of glucose concentrations.

The major problem with this system however is that it is not reversibleand incorporating a reducing agent into the sensor has been found to beimpractical. In order to be useful as a long term transdermal sensor,the chemical amplification process must be reversible. Severalcandidates for reversibility are available. For example, a novelruthenium porphyrin complex (e.g., RuO₂) may be included to catalyze thedecomposition of the dimer after it is formed. The use of this coupledreaction scheme means that the system is truly reagentless and itslifetime in the body is limited by the physiological response to theimplant.

HPAA may be modified (e.g., by diazotization) to produce a fluorescentproduct that is excitable at longer wavelengths. Alternatively, HPAA maybe modified to form a highly fluorescent but short-lived reactionproduct. This would involve creating a conformationally strainedintermediate such as the norbornyl cation attached to the basic HPAAbackbone. Two related approaches are also possible, which can beclassified as either fluorescent quenching or fluorescent enhancementapproaches. In the first case, the substrate of HRP is fluorescent andthe H₂O₂ produced by the GOX quenches this fluorescence by reacting withthe substrate bound to the HRP. In the second case, the H₂O₂ chemicallyoxidizes a non-fluorescent substrate bound to the HRP that fluoresceswhen oxidized. H₂O₂ can be used to oxidize a substrate that changedcolor. As an example, the type of chemistry used on the reflectancestrip could be immobilized on a gel and used in the transmission made ina finger web.

In addition to the fluorescence methods which can detect H₂O₂ (formedfrom the reaction shown in FIG. 7), there are two other possibilitiesthat can be used to detect H₂O₂. The first is the chemiluminescence ofluminol. Luminol upon oxidation by H₂O₂ undergoes chemiluminescence,where the intensity of the emitted light is proportional to the H₂O₂concentration. The second method is to use a dye such as3-methyl-2-benzothiazolinone hydrazone hydrochloride which turns a deepblue color in the presence of H₂O₂.

2. Equilibrium Binding Methods

Non-enzymatic equilibrium-based amplification methods forpolyhydroxylated analyte (e.g., glucose) amplification are preferable toenzymatic ones, because the ability of an enzyme to maintain itsactivity over long periods of time in the body is limited. In addition,enzymatic approaches based on O₂ consumption (for glucose measurement)suffer from the inherent deficiency of O₂ vs. glucose in the body andrequire a differentially permeable outer membrane.

Non-enzymatic equilibrium based amplification methods can be basedeither on lectins or on boronate (germinate or arsenate) based aromaticcompounds. Chick, U.S. Pat. No. 5,342,789 describes a competitivebinding approach whereas the present invention uses the simpler approachof attenuation in the fluorescence intensity of labeled lectinmolecules. One method utilizes a lectin such as concanavalin A (JackBean), Vicia faba (Fava Bean) or Vicia sativa. Such lectins bind glucosewith equilibrium constants of approximately 100. See, Falasca, et al.,Biochim. Biophys. Acta., 577:71 (1979). Labeling of the lectin with afluorescent moiety such as fluorescein isothiocyanate or rhodamine isrelatively straightforward using commercially available kits. FIG. 9shows the fluorescence spectrum of the rhodamine labeled concanavalin Ain different glucose concentrations. The mechanism of action of thelectin fluorescence quenching is presumably due to changes in themolecular conformation of the glucose containing lectin to that withoutthe glucose present. In the case of lectins, fluorescence quenching of afluorescein or rhodamine label occurs via an unknown mechanism, butpossibly due to the conformational change. Details for theimmobilization of rhodamine labeled lectin (concanavalin A) into apolyurethane (Jeffamine/Silicone polyurethane) membrane are providedbelow. The fluorescence of the labeled lectin decreased with increasingglucose concentration.

Another equilibrium binding approach to a single substrate system thatdoes not involve biomolecules is to use boronate based sugar bindingcompounds. The basic interaction between a sugar such as glucose and alabeled boronate complex is shown in FIG. 10. The binding of glucose tothe boronate group is reversible as shown in FIG. 10. In one case, thefluorescence of the boronate compounds is changed upon addition ofglucose. In other cases, fluorescence enhancement or quenching occursdue to intramolecular electron transfer. See, Falasca, et al., Biochim.Biophys. Acta., 577:71 (1979); Nakashima and Shinkai, Chemistry Letters,1267 (1994); and James, et al., J. Chem. Soc. Chem. Commun., 277 (1994).In some boronate complexes, modification of the acidity of the Lewisacid boron center is changed upon glucose binding.

Boronate complexes have been described which transduce a glucose signalthrough a variety of means. See, Nakashima, et al., Chem. Lett. 1267(1994); James, et al., J. Chem. Soc. Chem. Commun, 477 (1994); andJames, et al., Nature, 374:345 (1995). These include geometrical changesin porphyrin or indole type molecules, changes in optical rotation powerin porphyrins, and photoinduced electron transfer in anthracene typemoieties. Similarly, the fluorescence of 1-anthrylboronic acid has beenshown to be quenched by the addition of glucose. See, Yoon, et al., J.Am. Chem. Soc., 114:5874 (1992). A postulated mechanism for this effectis that of a shift in the Lewis acidity of the boronate group uponcomplexation of a diol. All these published approaches describe signaltransduction systems only.

An application to actual in vivo sensing for the above approaches mustalso encompass the immobilization of the transduction system into asuitable polymer system, which is preferably a biocompatible polymer. Inthe present invention, the transduction system, or signal amplificationcomponents are entrapped within a suitable polymer matrix.Alternatively, the amplification components can be covalently attachedto, and surrounded by the polymer matrix. Covalent attachment of thecomponents to a polymer matrix prevents leakage of the components tosurrounding tissue, and other undesirable contact of the amplificationcomponents with non-target fluids.

In one group of embodiments, the amplification components comprise anarylboronic acid moiety attached to an amine-functionalized dyemolecule. The linkage between the arylboronic acid moiety and the dyemolecule will typically be from about two to about four carbon atoms,preferably interrupted by one or more heteroatoms such as oxygen,sulfur, phosphorus or nitrogen. Certain non-limiting examples ofsuitable linkages include —CH₂—NH—CH₂—, —(CH₂)₂—NH—CH₂—,—C(O)CH₂—NH—CH₂—, —CH₂—NR—CH₂—, —(CH₂)₂—NR—CH₂—, and —C(O)CH₂—NR—CH₂—,in which the R group is an alkyl substituent of from 1 to about 8 carbonatoms. As used herein the term “alkyl” refers to a saturated hydrocarbonradical which may be straight-chain or branched-chain (for example,ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). This definition appliesboth when the term is used alone and when it is used as part of acompound term, such as “haloalkyl” and similar terms. Preferred alkylgroups are those containing 1 to 6 carbon atoms. All numerical ranges inthis specification and claims are intended to be inclusive of theirupper and lower limits. Additionally, the alkyl group which is attachedto a nitrogen atom in the linkages above will preferably be substitutedwith a functional group such as hydroxy, amino or thiol which willfacilitate the covalent attachment of the amplification component to abiocompatible matrix.

In a related group of embodiments, the implantable amplification systemincorporates a compound of the formula:

In this formula, D¹ represents a dye which can be a fluorescent dye, aluminescent dye or colorimetric dye. The symbols R¹, R³ and R⁴ eachindependently represent substituents which alter the electronicproperties of the groups to which they are attached or which containfunctional groups capable of forming covalent linkages to thesurrounding polymer matrix. Preferably, R¹, R³ and R⁴ are eachindependently hydrogen, hydroxy, acyl, C₁-C₄ alkoxy, halogen, thiol,sulfonic acid, sulfonamide, sulfinic acid, nitro, cyano, carboxylicacid, a C₁-C₁₂ alkyl group, a substituted C₁-C₁₂ alkyl group, a C₁-C₁₂alkenyl group, a substituted C₁-C₁₂ alkenyl group, a C₁-C₁₂ alkynylgroup, a substituted C₁-C₁₂ alkynyl group, aryl, substituted aryl,arylalkyl, substituted arylalkyl, amine, or substituted amine. For eachof the substituted species above, the substituents are preferablyhydroxy, acyl, aryl, C₁-C₄ alkoxy, halogen, thiol, sulfonic acid,amines, sulfonamide, sulfinic acid, nitro, cyano, carboxamide orcarboxylic acid. In particularly preferred embodiments, R¹, R³ and R⁴are each independently hydrogen, hydroxy, C₁-C₄ acyl, C₁-C₄ alkoxy,halogen, thiol, sulfonic acid, sulfonamide, nitro, cyano, carboxylicacid, a C₁-C₄ alkyl group, a C₁-C₄ alkenyl group, a C₁-C₄ alkynyl group,aryl, arylalkyl, or amine.

Each of the R² symbols independently represents hydrogen or C₁-C₄ alkyl,or taken together the two R² groups form a C₂-C₅ alkylene chain.Preferably, the R² groups are both hydrogen.

Each of L¹ and L² independently represent a linking group having fromzero to four contiguous atoms, preferably one to two. The linking groupsare preferably alkylene chains (e.g., methylene, ethylene, propylene, orbutylene). Alternatively, the alkylene chains can have one or more ofthe carbon atoms replaced by a oxygen, nitrogen, sulfur or phosphorus,with the understanding that any remaining valences on the heteroatomscan be occupied by hydrogen, hydroxy groups or oxo groups. Preferably,the heteroatoms when present, are oxygen or nitrogen.

The symbol Z represents a nitrogen, sulfur, oxygen or phosphorus. One ofskill would understand that for those embodiments in which Z is oxygen,R¹ will not be present. Additionally, as above, any remaining valenceson the heteroatoms can be occupied by hydrogen, hydroxy groups or oxogroups. Preferably, Z is nitrogen. The symbol x is an integer of fromzero to four.

The chemical terms used herein are taken to have their accepted meaningsto one of skill in the chemical arts. For example, the term “alkoxy”refers to an alkyl radical as described above which also bears an oxygensubstituent which is capable of covalent attachment to anotherhydrocarbon radical (such as, for example, methoxy, ethoxy, andt-butoxy). “Halogen” is meant to include —F, —Cl, —Br and —I, although—F and —Cl are preferred. The term “alkenyl” as used herein refers to analkyl group as described above which contains one or more sites ofunsaturation. The term “alkynyl” as used herein refers to an alkyl groupas described above which contains one or more carbon-carbon triplebonds. The term “aryl” refers to an aromatic substituent which may be asingle ring or multiple rings which are fused together, linkedcovalently or linked to a common group such as an ethylene or methylenemoiety. The aromatic rings may each contain heteroatoms, for example,phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl,thienyl, pyridyl and quinoxalyl. The aryl moieties may also beoptionally substituted as discussed above. Additionally, the arylradicals may be attached to other moieties at any position on the arylradical which would otherwise be occupied by a hydrogen atom (such as,for example, 2-pyridyl, 3-pyridyl and 4-pyridyl). The term “arylalkyl”refers to an aryl radical attached directly to an alkyl group.

Preferably, the dye used in formula (I) is an anthracene, fluorescein,xanthene (e.g., sulforhodamine, rhodamine), cyanine, coumarin (e.g.,coumarin 153), oxazine (e.g., Nile blue), a metal complex or otherpolyaromatic hydrocarbon which produces a fluorescent signal. Structuresfor some of the embodiments of formula I are provided in FIG. 11 alongwith the excitation and emission wavelengths for each. Particularlypreferred are long wavelength fluorescent dyes having emissionwavelengths of at least about 450 nm, preferably from 450 to about 800nm. Shorter wavelength dyes typically do not provide sufficient signalthrough the skin. As a result, shorter wavelength dyes are suitable forapplications in which interrogation and signal delivery is by means of afiber optic. Preferred shorter wavelength dyes are those having emissionwavelengths of about 320 nm to about 450 nm.

The compounds used in this aspect of the invention can be prepared bythe methods described in the examples below or by modifications thereof.FIG. 12 provides one synthesis scheme for the compounds of formula I. Inthis scheme, 9-anthraldehyde (available from commercial sources such asAldrich Chemical Co., Milwaukee, Wis., USA) can be treated with5-amino-1-pentanol (Aldrich) under reductive amination conditions usingsodium borohydride in methanol. The resulting secondary amine can thenbe alkylated with a bromomethyl arylboronic acid derivative to provide aprotected amplification component.

FIG. 13 provides another synthesis scheme for the compounds of formulaI. In this scheme, 10-(hydroxymethyl)-9-anthraldehyde (preparedaccording to the methods described in Lin, et al., J. Org. Chem. 44:4701(1979)) is reductively aminated using methylamine in a two-step processinvolving imine formation followed by sodium borohydride reduction ofthe imine. Alkylation of the secondary amine with a suitable arylboronicacid derivative then provides the desired compound of formula I. In thisfamily of compounds, the D¹ moiety (e.g., anthracene) has an attachedhydroxymethyl group which facilitates covalent attachment of thecompound to a biocompatible matrix.

3. Spectroscopic Method

Another approach to minimally invasive glucose sensing is by surfaceenhanced resonance Raman spectroscopy. The glucose is bound to asubstrate like concanavalin A or a boric acid complex and the Ramanspectrum measured.

Immobilization of the Amplification Components in a Polymer Matrix

In order to use the amplification components for analyte sensing invivo, the components for the reactions must be immobilized in a polymermatrix that can be implanted subdermally. The matrix should be permeableto the analyte of interest and be stable within the body. Still further,the matrix should be prepared from biocompatible materials, oralternatively, coated with a biocompatible polymer. As used herein, theterm “biocompatible” refers to a property of materials or matrix whichproduce no detectable adverse conditions upon implantation into ananimal. While some inflammation may occur upon initial introduction ofthe implantable amplification system into a subject, the inflammationwill not persist and the implant will not be rendered inoperable byencapsulation (e.g., scar tissue).

The biocompatible matrix can include either a liquid substrate (e.g., acoated dialysis tube) or a solid substrate (e.g.,polyurethanes/polyureas, silicon-containing polymers, hydrogels, solgelsand the like). Additionally, the matrix can include a biocompatibleshell prepared from, for example, dialysis fibers, teflon cloth,resorbable polymers or islet encapsulation materials. The matrix can bein the form of a disk, cylinder, patch, microspheres or a refillablesack and, as noted, can further incorporate a biocompatible mesh thatallows for full tissue ingrowth with vascularization. While subdermalimplantation is preferred, one skilled in the art would realize otherimplementation methods could be used. The key property of the matrix isits permeability to analytes and other reactants necessary for chemicalamplification of a signal. For example, a glucose monitoring matrix mustbe permeable to glucose. In the case of the enzymatic approach, thematrix must also be permeable to O₂ and be compatible with H₂O₂. Whileoxygen and glucose permeability are required to form the H₂O₂, hydrogenperoxide permeability is necessary for the optical sensor. Finally, theimplant should be optically transparent to the light from the opticalsource used for interrogating the IAS.

FIG. 14 provides an illustration of several embodiments. As seen in FIG.14A, an amplification system can encompass a substrate layer, atransducer layer containing the amplification components, and a layerwhich is permeable to the analyte of interest.

The substrate layer can be prepared from a polymer such as apolyurethane, silicone, silicon-containing polymer, chronoflex, P-HEMAor sol-gel. The substrate layer can be permeable to the analyte ofinterest, or it can be impermeable. For those embodiments in which thesubstrate layer is impermeable, the amplification components will becoated on the exterior of the substrate layer and further coated with apermeable layer (see FIG. 14A).

In some embodiments, the amplification components will be entrapped orencased via covalent attachment, within a matrix which is itselfpermeable to the analyte of interest and biocompatible (see FIG. 14B).In these embodiments, a second permeable layer is unnecessary.Nevertheless, the use of a permeable layer such as a hydrogel whichfurther facilitates tissue implantation is preferred (see FIG. 14C).

1. Biocomnatible Matrix Materials

For those embodiments in which a polymer matrix is to be placed incontact with a tissue or fluid, the polymer matrix will preferably be abiocompatible matrix. In addition to being biocompatible, anotherrequirement for this outermost layer of an implantable amplificationsystem is that it be permeable to the analyte of interest. A number ofbiocompatible polymers are known, including some recently describedsilicon-containing polymers (see application Ser. No. 08/721,262, filedSep. 26, 1996, now U.S. Pat. No. 5,777,060 and incorporated herein byreference) and hydrogels (see U.S. Pat. No. 08,749,754, filed Oct. 24,1996, now U.S. Pat. No. 5,786, 439 and incorporated herein byreference). Silicone-containing polyurethane can be used for theimmobilization of most of the glucose binding systems or other analyteamplification components. Other polymers such as silicone rubbers (NuSil4550), biostable polyurethanes (Biomer, Tecothane, Tecoflex, Pellethaneand others), PEEK (polyether ether ketone) acrylics or combinations arealso suitable.

a. Silicon-Containing Polymers

In one group of embodiments, the amplification components are eitherentrapped in, or covalently attached to a silicone-containing polymer.This polymer is a homogeneous matrix prepared from biologicallyacceptable polymers whose hydrophobic/hydrophilic balance can be variedover a wide range to control the rate of polyhydroxylated analytediffusion to the amplification components. The matrix can be prepared byconventional methods by the polymerization of diisocyanates, hydrophilicdiols or diamines, silicone polymers and optionally, chain extenders.The resulting polymers are soluble in solvents such as acetone orethanol and may be formed as a matrix from solution by dip, spray orspin coating. Preparation of biocompatible matrices for glucosemonitoring have been described in application Ser No. 08/721,262, nowU.S. Pat. Nos. 5,777,060, and 08/749,754, now U.S. Pat. No. 5,786,439,the disclosures of which have previously been incorporated herein byreference.

The diisocyanates which are useful for the construction of abiocompatible matrix are those which are typically those which are usedin the preparation of biocompatible polyurethanes. Such diisocyanatesare described in detail in Szycher, SEMINAR ON ADVANCES IN MEDICAL GRADEPOLYURETHANES, Technomic Publishing, (1995) and include both aromaticand aliphatic diisocyanates. Examples of suitable aromatic diisocyanatesinclude toluene diisocyanate, 4,4′-diphenylmethane diisocyanate,3,3′-dimethyl-4,4′-biphenyl diisocyanate, naphthalene diisocyanate andparaphenylene diisocyanate. Suitable aliphatic diisocyanates include,for example, 1,6-hexamethylene diisocyanate (HDI),trimethylhexamethylene diisocyanate (TMDI), trans-1,4-cyclohexanediisocyanate (CHDI), 1,4-cyclohexane bis(methylene isocyanate) (BDI),1,3-cyclohexane bis(methylene isocyanate) (H₆XDI), isophoronediisocyanate (IPDI) and 4,4′-methylenebis(cyclohexyl isocyanate)(H₁₂MDI). In preferred embodiments, the diisocyanate is isophoronediisocyanate, 1,6-hexamethylene diisocyanate, or4,4′-methylenebis(cycloheyl isocyanate). A number of these diisocyanatesare available from commercial sources such as Aldrich Chemical Company(Milwaukee, Wis., USA) or can be readily prepared by standard syntheticmethods using literature procedures.

The quantity of diisocyanate used in the reaction mixture for thepresent compositions is typically about 50 mol % relative to thecombination of the remaining reactants. More particularly, the quantityof diisocyanate employed in the preparation of the present compositionswill be sufficient to provide at least about 100% of the —NCO groupsnecessary to react with the hydroxyl or amino groups of the remainingreactants. For example, a polymer which is prepared using x moles ofdiisocyanate, will use a moles of a hydrophilic polymer (diol, diamineor combination), b moles of a silicone polymer having functionalizedtermini, and c moles of a chain extender, such that x=a+b+c, with theunderstanding that c can be zero.

A second reactant used in the preparation of the biocompatible matrixdescribed herein is a hydrophilic polymer. The hydrophilic polymer canbe a hydrophilic diol, a hydrophilic diamine or a combination thereof.The hydrophilic diol can be a poly(alkylene)glycol, a polyester-basedpolyol, or a polycarbonate polyol. As used herein, the term“poly(alkylene)glycol” refers to polymers of lower alkylene glycols suchas poly(ethylene)glycol, poly(propylene)glycol and polytetramethyleneether glycol (PTMEG). The term “polycarbonate polyol” refers thosepolymers having hydroxyl functionality at the chain termini and etherand carbonate functionality within the polymer chain. The alkyl portionof the polymer will typically be composed of C2 to C4 aliphaticradicals, or in some embodiments, longer chain aliphatic radicals,cycloaliphatic radicals or aromatic radicals. The term “hydrophilicdiamines” refers to any of the above hydrophilic diols in which theterminal hydroxyl groups have been replaced by reactive amine groups orin which the terminal hydroxyl groups have been derivatized to producean extended chain having terminal amine groups. For example, a preferredhydrophilic diamine is a “diamino poly(oxyalkylene)” which ispoly(alkylene)glycol in which the terminal hydroxyl groups are replacedwith amino groups. The term “diamino poly(oxyalkylene” also refers topoly(alkylene)glycols which have aminoalkyl ether groups at the chaintermini. One example of a suitable diamino poly(oxyalkylene) ispoly(propylene glycol)bis(2-aminopropyl ether). A number of the abovepolymers can be obtained from Aldrich Chemical Company. Alternatively,literature methods can be employed for their synthesis.

The amount of hydrophilic polymer which is used in the presentcompositions will typically be about 10% to about 80% by mole relativeto the diisocyanate which is used. Preferably, the amount is from about20% to about 60% by mole relative to the diisocyanate. When loweramounts of hydrophilic polymer are used, it is preferable to include achain extender (see below).

Silicone polymers which are useful for the determination ofpolyhydroxylated analytes (e.g., glucose) are typically linear. Forpolymers useful in glucose monitoring, excellent oxygen permeability andlow glucose permeability is preferred. A particularly useful siliconepolymer is a polydimethylsiloxane having two reactive functional groups(i. e, a functionality of 2). The functional groups can be, for example,hydroxyl groups, amino groups or carboxylic acid groups, but arepreferably hydroxyl or amino groups. In some embodiments, combinationsof silicone polymers can be used in which a first portion compriseshydroxyl groups and a second portion comprises amino groups. Preferably,the functional groups are positioned at the chain termini of thesilicone polymer. A number of suitable silicone polymers arecommercially available from such sources as Dow Chemical Company(Midland, Mich., USA) and General Electric Company (Silicones Division,Schenectady, N.Y., USA). Still others can be prepared by generalsynthetic methods known to those skilled in the art, beginning withcommercially available siloxanes (United Chemical Technologies, Bristol,Pa., USA). For use in the present invention, the silicone polymers willpreferably be those having a molecular weight of from about 400 to about10,000, more preferably those having a molecular weight of from about2000 to about 4000. The amount of silicone polymer which is incorporatedinto the reaction mixture will depend on the desired characteristics ofthe resulting polymer from which the biocompatible membrane are formed.For those compositions in which a lower analyte penetration is desired,a larger amount of silicone polymer can be employed. Alternatively, forcompositions in which a higher analyte penetration is desired, smalleramounts of silicone polymer can be employed. Typically, for a glucosesensor, the amount of siloxane polymer will be from 10% to 90% by molerelative to the diisocyanate. Preferably, the amount is from about 20%to 60% by mole relative to the diisocyanate.

In one group of embodiments, the reaction mixture for the preparation ofbiocompatible membranes will also contain a chain extender which is analiphatic or aromatic diol, an aliphatic or aromatic diamine,alkanolamine, or combinations thereof. Examples of suitable aliphaticchain extenders include ethylene glycol, propylene glycol,1,4-butanediol, 1,6-hexanediol, ethanolamine, ethylene diamine, butanediamine, 1,4-cyclohexanedimethanol. Aromatic chain extenders include,for example, para-di(2-hydroxyethoxy)benzene,meta-di(2-hydroxyethoxy)benzene, Ethacure 100® (a mixture of two isomersof 2,4-diamino-3,5-diethyltoluene), Ethacure 300®(2,4-diamino-3,5-di(methylthio)toluene),3,3′-dichloro4,4′diaminodiphenylmethane, Polacure® 740 M (trimethyleneglycol bis(para-aminobenzoate)ester), and methylenedianiline.Incorporation of one or more of the above chain extenders typicallyprovides the resulting biocompatible membrane with additional physicalstrength, but does not substantially increase the glucose permeabilityof the polymer. Preferably, a chain extender is used when lower (i.e.,10-40 mol %) amounts of hydrophilic polymers are used. In particularlypreferred compositions, the chain extender is diethylene glycol which ispresent in from about 40% to 60% by mole relative to the diisocyanate.

b. Hydrogels

In some embodiments, the polymer matrix containing the amplificationcomponents can be further coated with a permeable layer such as ahydrogel, cellulose acetate, P-HEMA, nafion, or glutaraldehyde. A numberof hydrogels are useful in the present invention. For those embodimentsin which glucose monitoring is to be conducted, the preferred hydrogelsare those which have been described in U.S. Ser. No. 08/749,754, nowU.S. Pat. No. 5,786,439the disclosure of which has previously beenincorporated herein by reference. Alternatively, hydrogels can be usedas the polymer matrix which encase or entrap the amplificationcomponents. In still other embodiments, the amplification components canbe covalently attached to a hydrogel.

Suitable hydrogels can be prepared from the reaction of a diisocyanateand a hydrophilic polymer, and optionally, a chain extender. Thehydrogels are extremely hydrophilic and will have a water pickup of fromabout 120% to about 400% by weight, more preferably from about 150% toabout 400%. The diisocyanates, hydrophilic polymers and chain extenderswhich are used in this aspect of the invention are those which aredescribed above. The quantity of diisocyanate used in the reactionmixture for the present compositions is typically about 50 mol %relative to the combination of the remaining reactants. Moreparticularly, the quantity of diisocyanate employed in the preparationof the present compositions will be sufficient to provide at least about100% of the —NCO groups necessary to react with the hydroxyl or aminogroups of the remaining reactants. For example, a polymer which isprepared using x moles of diisocyanate, will use a moles of ahydrophilic polymer (diol, diamine or combination), and b moles of achain extender, such that x=a+b, with the understanding that b can bezero. Preferably, the hydrophilic diamine is a “diaminopoly(oxyalkylene)” which is poly(alkylene)glycol in which the terminalhydroxyl groups are replaced with amino groups. The term “diaminopoly(oxyalkylene” also refers to poly(alkylene)glycols which haveaminoalkyl ether groups at the chain termini. One example of a suitablediamino poly(oxyalkylene) is poly(propylene glycol) bis(2-aminopropylether). A number of diamino poly(oxyalkylenes) are available havingdifferent average molecular weights and are sold as Jeffamines® (forexample, Jeffamine 230, Jeffamine 600, Jeffamine 900 and Jeffamine2000). These polymers can be obtained from Aldrich Chemical Company.Alternatively, literature methods can be employed for their synthesis.

The amount of hydphlic polymer which is used in the present compositionswill typically be about 10% to about 100% by mole relative to thediisocyanate which is used. Preferably, the amount is from about 50% toabout 90% by mole relative to the diisocyanate. When amounts less than100% of hydrophilic polymer are used, the remaining percentage (to bringthe total to 100%) will be a chain extender.

Polymerization of the substrate layer components or the hydrogelcomponents can be carried out by bulk polymerization or solutionpolymerization. Use of a catalyst is preferred, though not required.Suitable catalysts include dibutyltin bis(2-ethylhexanoate), dibutyltindiacetate, triethylamine and combinations thereof. Preferably dibutyltinbis(2-ethylhexanoate is used as the catalyst. Bulk polymerization istypically carried out at an initial temperature of about 25° C. (ambienttemperature) to about 50° C., in order to insure adequate mixing of thereactants. Upon mixing of the reactants, an exotherm is typicallyobserved, with the temperature rising to about 90-120° C. After theinitial exotherm, the reaction flask can be heated at from 75° C. to125° C., with 90° C. to 100° C. being a preferred temperature range.Heating is usually carried out for one to two hours.

Solution polymerization can be carried out in a similar manner. Solventswhich are suitable for solution polymerization include, tetrahydrofuran,dimethylformamide, dimethyl sulfoxide, dimethylacetamide, halogenatedsolvents such as 1,2,3-trichloropropane, and ketones such as4-methyl-2-pentanone. Preferably, THF is used as the solvent. Whenpolymerization is carried out in a solvent, heating of the reactionmixture is typically carried out for at least three to four hours, andpreferably at least 10-20 hours. At the end of this time period, thesolution polymer is typically cooled to room temperature and poured intoDI water. The precipitated polymer is collected, dried, washed with hotDI water to remove solvent and unreacted monomers, then re-dried.

2. Methods for Immobilizing the Amplification Components

Immobilization of the amplification components into a polymer matrixdescribed above can be accomplished by incorporating the components intothe polymerization mixture during formation of the matrix. If thecomponents are prepared having suitable available functional groups thecomponents will become covalently attached to the polymer duringformation. Alternatively, the components can be entrapped within thematrix during formation.

a. Covalent Attachment

In one group of embodiments, the enzymes and substrates of thefluorescence generating reaction are immobilized in, or on the surface,of an appropriate base material using covalent bonding chemistry. Theenzymes can be bonded to one component of the base polymer using any ofa variety of covalent bonding techniques such as streptavidin/biotincoupling. The substrates of the fluorescence generating reaction can becovalently bonded to the base material using any of a variety ofcovalent bonding techniques commonly used for polymer synthesis, forexample, via condensation, condensation-elimination or free radicalpolymerizations. For compounds of formula I, the appropriatefunctionalization could be accomplished at one or more of the pendantgroups R¹, R³ or R⁴. For condensation type polymerizations, the use of asingle covalent linker would lead to a terminal attachment, whereas theuse of two or three R groups leads to chain extension and crosslinking,respectively. For free radical type polymerizations, chain extension canoccur with a single functionalized R group.

As outlined in Example 3, an amine-terminated block copolymer,poly(propylene glycol)-block-poly(ethylene glycol)-block-poly(propyleneglycol)bis(2-aminopropyl ether), can be reacted with a diisocyanate toform a biocompatible hydrophilic polyurea. Incorporation of a hydroxyfunctionalized fluorescent monomer provides a polymer containing acovalently attached amplification component, in this example, as a chainterminating urethane linkage. In any case, the goal of immobilization isto incorporate the amplification components into a matrix in such a wayas to retain the molecular system's desired optical and chemicalactivity. FIG. 16 shows the reversible change in fluorescence for asolution of anthracene-boronate as a function of glucose concentrationover the physiological range.

In some embodiments, the amplification components will not besubstituted with suitable functional groups for covalent attachment to apolymer during formation. In this instance, the reagents are simplyentrapped. The amount of amplification component used for either thecovalent or entrapped methods will typically be on the order of about0.5% to about 10% by weight, relative to the total weight of thebiocompatible matrix. One of skill in the art will understand that theamounts can be further adjusted upward or downward depending on theintensity of the signal produced as well as the sensitivity of thedetector.

Optical Systems

The second aspect of the biosensors described herein consists of anoptical system for interrogating the IAS and detecting the signal thusproduced by the IAS. As used herein, the term “interrogating” refers toillumination of the amplification components in the IAS and subsequentdetection of the emitted light. One embodiment illustrating atransdermal optical system is shown in FIG. 1, where the light source(S) shines through the skin, and a detector (D) detects the fluorescencetransmitted through the skin. FIGS. 3-6 show embodiments where there isno transmission through the skin, as the light source is implanted orthe light travels via a fiber optic to the amplification systempositioned at the end of the fiber.

FIG. 1 shows a schematic of the subdermally implanted optical glucosemonitoring system. The light source (S) could be a lamp, an LED, or alaser diode (pulsed or modulated). The detector (D) can be a photodiode,CCD detector or photomultiplier tube. Optionally, filters are used tofilter the incident and/or emitted beams of light to obtain desiredwavelengths. The source and detector are shown in FIG. 1 as positionedoutside the body, although the source and/or the detector can beimplanted as shown in FIGS. 3-6. The biocompatible material (e.g.,silicone, polyurethane or other polymer) with the immobilizedamplification components is implanted under the skin. The light sourceis used to illuminate the implanted system, and the detector detects theintensity of the emitted (typically fluorescent) light. Other modes ofinteraction may also be used, such as absorbance, transmittance, orreflectance, when the change in the amount of light or spectralcharacter of the light that is measured by the detector or spectrometeris modulated by the local analyte (e.g., glucose) concentration. In yetother detection methods, the fluorescence lifetimes are measured ratherthan the light intensity.

In the case of fluorescence, the ratio of intensity of excitation andemission can be used to quantify the glucose signal. In a preferredembodiment, the ratio of fluorescence from the amplification componentsto the fluorescence of a calibration fluorophore is measured. Thismethod eliminates errors due to registration and variations of lighttransport through the skin (e.g., caused by different skin tones).

Methods for the Detection and Quantitation of Analytes In Vivo

In view of the above compositions and devices, the present inventionalso provides methods for the detection and quantitation of an analytein vivo. More particularly, the methods involve quantifying the amountof a polyhydroxylated analyte in an individual, by

(a) interrogating a subcutaneously implanted amplification system withan energy source to provide an excited amplification system whichproduces an energy emission corresponding to the amount of thepolyhydroxylated analyte; and

(b) detecting the emission to thereby quantify the amount of thepolyhydroxylated analyte in the individual.

The amplification and optical systems are essentially those which havebeen described above, and the preferred embodiments including componentsof the biocompatible matrix (e.g., silicon-containing polymers,hydrogels, etc.) are also those which have been described above. Priorto carrying out the present method, the amplification system isimplanted in an individual using minimally invasive surgical ormicrosurgical techniques. The purpose of such implantation is to placein contact the amplification system and the analyte of interest (e.g.,in fluid or tissue containing the analyte). Accordingly, theamplification system can be placed in or under the skin, oralternatively within an organ or blood vessel. When transdermalinterrogation is used, the amplification system is preferably placedsubcutaneously about 1-2 mm below the skin surface. For fiber opticmediated interrogation, the depth will be from 1-4 mm below the skinsurface. For those embodiments in which the optical system andamplification components are in communication with an insulin pump, theplacement can be at even greater depths.

The polyhydroxylated analyte can be any of a variety of endogenous orxenobiotic substances which have two or more hydroxy functional groupsin positions vicinal to each other. Preferably, the analyte is a sugar,more preferably glucose.

As already noted, suitable amplification systems have been describedabove. However, in certain preferred embodiments, the implantedamplification system will further comprise a calibration fluorophorewhich provides a signal not interfering with the signal from theamplification components. In some preferred embodiments, the IAScomprises a boronate based sugar binding compound, more preferably thoseof formula I and a calibration fluorophore. Suitable calibrationfluorophores are those fluorescent dyes such as fluoresceins, coumarins,oxazines, xanthenes, cyanines, metal complexes and polyaromatichydrocarbons which produce a fluorescent signal. In other preferredembodiments, the amplification system will comprise a calibrationfluorophore and a compound of formula I in which D¹ is a long wavelengthfluorescent dye.

In order that those skilled in the art can more fully understand thisinvention, the following examples illustrating the general principlesfor preparation of glucose responsive systems are set forth. Theseexamples are given solely for purposes of illustration, and should notbe considered as expressing limitations unless so set forth in theappended claims. All parts are percentages by weight, unless otherwisestated.

EXAMPLES

In the examples below, Example 1 provides the synthesis of variouschemical amplification components. Example 2 provides the synthesis ofbiocompatible polymers. Example 3 provides a description of the covalentattachment of certain amplification components to a biocompatiblepolymer.

General Materials and Methods

Unless otherwise noted, the materials used in the examples were obtainedfrom Aldrich Chemical Co., Milwaukee, Wisc., USA or Sigma ChemicalCompany, St. Louis, Mo., USA.

Example 1

1.1 Production of para-Hydroxyphenylacetic Acid Dimer

A solution of glucose oxidase (10 U/ml) (GOX), horseradish peroxidase (1U/ml) (HRP) and para-hydroxyphenylacetic acid were mixed in a cuvetteinside a spectrofluorimeter. At time 0, an aliquot of glucose (100mg/dl) was added and the fluorescence intensity was monitored as afunction of time. FIG. 8 shows the calibration curve of glucoseconcentration vs. fluorescence intensity.

1.2 Synthesis of Fluorescein Labeled Boronic Acid (FABA)

Preparation of a fluorescein labeled boronic acid (FABA) was carried outas described in Uziel, et al., Biochem. Biophys. Res. Commun., 180:1233(1991), incorporated herein by reference.

Briefly, a solution (5 mL) of 3-aminophenylboronic acid was prepared inDI water. The pH was adjusted to 8 with NaOH and NaHCO₃. Fluoresceinisothiocyanate (0.45 mmol) was added and the mixture was stirredovernight at room temperature. The fluorescein-labeled boronate wasisolated as yellow crystals. At pH 10, the fluorescence of the compoundwas significantly decreased by the addition of glucose to the solution.

1.3 Synthesis of Labeled Boronic Acids

The labeled boronic acids described herein are prepared as outlined inthe scheme depicted in FIGS. 12 and 13.

2,4,6-(o-(bromomethyl)phenyl)boroxin (1) was prepared according to aliterature procedure from 2,4,6-o-Tolylboroxin substitutingbenzoylperoxide (BPO) for the AIBN catalyst (see, Hawkins, et al., J.Am. Chem. Soc. 82:3863 (1960)).

9-((N-Methyl-N-(o-boronobenzyl)amino)methyl)anthracene (2) wassynthesized by a modification of a literature procedure (A)(see, James,et al., J. Am. Chem. Soc. 117:8982 (1995)) or by method (B).

(A): 2,4,6-(o-(bromomethyl)phenyl)boroxin (100 mg, 0.18 mmol) and9-((methylamino)methyl)anthracene (254 mg, 1.1 mmol) were refluxed in 50mL chloroform for 3 h. The mixture was cooled to 0° C. in an ice bathand filtered through a sintered glass frit. Solvent was removed from thefiltrate under reduced pressure. The crude material was washed with 3×3mL portions of acetonitrile/water (9/1, v/v) to remove the hydrochloridesalt of 9-((methylamino)methyl)anthracene to provide 2 as a pale yellowpowder: 155 mg (48%); mp 149-151° C. (lit. mp 147-152° C.); ¹H NMR(300.13 MHz, CD₃OD) δ2.27 (s, 3H), 3.85 (s, 2H), 4.60 (s, 2H), 7.00-7.80(m, 8H). 8.05 (m, 2H), 8.30-8.70 (m, 3H).

(B): A solution of 9-((methylamino)methyl)anthracene (1.00 g, 4.5 mmol),2-bromobenzyl bromide (1.13 g, 4.5 mmol) and K₂CO₃ (0.691 g, 5.0 mmol)in 50 mL of acetonitrile was refluxed under nitrogen for 18 hr. Thesolution was filtered on a sintered-glass filter and solvent was removedfrom the filtrate under reduced pressure to yield9-(N-Methyl-N-(o-bromobenzyl)amino)methyl)anthracene (95% by NMR). Theresulting solid was taken up in diethyl ether (50 mL), and treated at 0°C. with 1 equiv of butyllithium. The mixture was stirred at 0° C. for 2h at which time 5 equiv of trimethyborate was added via a cannula. Afterwarming the mixture to room temperature, 50 mL of water was added toquench the reaction. The ether layer was separated, washed with 3×10 mLwater, and dried over sodium sulfate. Removal of the solvent underreduced pressure provided a solid identical to that obtained in (A) in52% yield.

2,2-Dimethylpropane-1,3-diyl(o-(bromomethyl)phenyl)boronate (3)

2,4,6-(o-(bromomethyl)phenyllboroxin (5.0 g, 25.4 mmol) and2,2-dimethyl-1,3-propanediol (8.73 g, 83.8 mmol) were refluxed intoluene (200 mL) with azeotropic removal of water (Dean-Stark) for 24 h.The solvent was removed under vacuum to give a solid/oil mixture whichwas then slurried in 25 mL toluene and silica gel. The resulting mixturewas filtered on a sintered-glass frit and rinsed thoroughly with coldtoluene until the washings revealed no evidence of product by TLC. Thecombined filtrate was evaporated under reduced pressure to give 3 as apale yellow oil: 6.76 g (94%); ¹H NMR (300.13 MHz, CD₃CN) δ1.05 (s, 6H),3.81 (s, 4H), 4.95 (s, 2H), 7.15-7.45 (m, 3H), 7.74 (m, 1H).

9,10-Bis((Methylamino)methyl)anthracene (4)

The title compound was prepared according to a literature procedure(see, James, et al., J. Am. Chem. Soc. 117:8982 (1995)).

9-((5-hydroxypentyl)aminomethyl)anthracene (5)

A solution of anthraldehyde (9.595 g, 0.0465 mol) and 5-amino-1-pentanol(15.00 g, 0.116 mol) dissolved in 500 mL ethanol at 0° C. was stiffedfor 3 h. After warming to room temperature the solvent was removed underreduced pressure, and 150 mL of ethanol containing NaBH₄ (4.65 g, 0.1229mol) was added slowly with stirring. The resulting mixture was allowedto stir overnight. The ethanol was removed under reduced pressure and tothe brown oil/solid mixture was added 150 mL diethyl ether. Water wasadded dropwise to this solution until the evolution of hydrogen ceased,followed by the addition of 500 mL water. The ether phase was isolated,washed with 2×50 mL water, dried over sodium sulfate and filtered on asintered-glass frit. Removal of the solvent afforded 12.17 g (89.2%yield) of 5 as a golden solid; ¹H NMR (300.13 MHz, CD₃CN) δ1.51 (m, 6H),2.81 (t, 2H), 3.49 (t, 2H), 4.68 (s, 2H), 7.52 (m, 4H), 8.05 (d, 2H),8.44 (m, 3H); ¹³C-{¹H} NMR (75.4 MHz, CDCl₃) δ133.1, 132.0, 131.7,130.4, 128.6, 127.4, 126.2, 125.1, 62.9, 51.1, 45.8, 33.5, 30.3, 24.8.

9-((N-(5-hydroxypentyl)-N-(ethyl)amino)methyl)anthracene (6)

9-((5-hydroxypentyl)aminomethyl)anthracene (1.00 g, 3.41 mol) and K₂CO₃(0.518 g, 3.75 mmol) were taken up in 25 mL acetonitrile. Ethyl bromide(11.14 g, 102 mmol) was added and the mixture was refluxed undernitrogen for 24 h. The mixture was filtered on a sintered-glass frit andthe solvent and excess ethyl bromide was removed under reduced pressure.Removal of the solvent afforded 1.07 g (98% yield) of 6 as a yellowsolid; ¹H NMR (300.13 MHz, CD₃CN) δ1.15 (m, 2H), 1.38 (m, 5H), 1.81 (m,2H), 3.05 (m, 4H), 3.49 (t, 2H), 5.21 (s, 2H), 7.45 (m, 2H), 7.62 (m,2H), 8.05 (d, 2H), 8.44 (m, 3H); ¹³C-{¹H} NMR (75.4 MHz, CDCl₃) δ131.8,131.3, 130.8, 129.6, 128.0, 125.6, 124.0, 61.7, 53.1, 49.4, 48.7, 31.4,23.9, 23.3, 10.0.

9-((N-(5-hydroxypentyl)-N-(o-boronobenzyl)amino)methyl)anthracene (7)

9-((5-hydroxypentyl)aminomethyl)anthracene (1.06 g, 3.51 mol) and K₂CO₃(0.56 g, 4.05 mmol) were taken up in 15 mL acetonitrile. A solution of2,2-dimethylpropane-1,3-diyl(o-)bromomethyl)phenyl)boronate (1.02 g,3.51 mmol) in 5 mL acetonitrile was added and the mixture was refluxedunder nitrogen for 24 h. The mixture was filtered on a sintered-glassfrit and the solvent was removed under reduced pressure. The resultingsolid was triturated with acetonitrile/water (4:1, v/v) to deprotect theboronate group, filtered on a sintered-glass frit and vacuum dried toyield 9 as a pale yellow solid (0.744 g, 48% yield); ¹H NMR (300.13 MHz,CD₃OD) δ0.95 (m, 2H), 1.15 (m, 2H), 1.60 (m, 2H), 2.82 (m, 2H), 3.45 (m,2H), 4.45 (s, 2H), 5.08 (s, 2H), 7.1-7.8 (m, 8H), 8.07 (d, 2H), 8.21 (d,2H), 8.62 (s, 1H); ¹³C-{¹H} NMR (75.4 MHz, CDCl₃) δ135.8, 133.0, 131.5,129.1, 128.6, 127.8, 126.5, 124.9, 62.4, 61.9, 53.7, 49.7, 32.6, 24.9,24.6.

10-(hydroxymethyl)-9-anthraldehyde (8) was prepared according to aliterature procedure (see, Lin, et al., J. Org. Chem. 44:4701 (1979)).

10-(hydroxymethyl)-9-((methylimino)methyl)anthracene (9)

10-(hydroxymethyl)-9-anthraldehyde (3.00 g, 12.7 mmol) was added to 50mL of a saturated solution of methylamine in methanol and stirred atroom temperature for 2 h. The solvent and excess methylamine was removedunder reduced pressure to afford the imine as a bright yellow powder(quant); ¹H NMR (300.13 MHz, DMSO-d₆) δ3.35 (s, 3H), 5.51 (s, 2H), 7.61(m, 4H), 8.55 (m, 4H), 9.48 (s, 1H).

10-(hydroxymethyl)-9-((methylamino)methyl)anthracene (10)

10-(hydroxymethyl)-9-((methylimino)methyl)anthracene (1.00 g, 4.0 mmol)was slurried in 25 mL isopropanol. NaBH₄ (0.454 g, 12.0 mmol) was addedas a solid and the solution was stirred at room temperature for 72 h.The mixture was filtered on a sintered-glass frit and the solvent wasremoved under reduced pressure to yield 10 as a bright yellow powder(0.853 g, 86% yield): ¹H NMR (300.13 MHz, CD₃OD) δ2.55 (s, 3H), 4.64 (s,2H), 5.56 (s, 2H), 7.55 (m, 4H), 8.38 (m, 2H), 8.50 (m, 2H); ¹³C-{¹H}NMR (75.4 MHz, CD₃OD) δ133.4, 133.0, 131.6, 131.5, 126.9, 126.7, 126.2,125.7, 57.2, 47.9, 36.5.

10-(hydroxymethyl)-9-N-(o-boronobenzyl)amino)methylanthracene (11)

10-(hydroxymethyl)-9-((methylamino)methyl)anthracene (0.800 g, 3.18mmol) and K₂CO₃ (0.56 g, 4.05 mmol) were taken up in 15 mL acetonitrile.A solution of2,2-dimethylpropane-1,3-diyl(o-(bromomethyl)phenyl)boronate 3 (1.00 g,3.44 mmol) in 5 mL acetonitrile was added and the mixture was refluxedunder nitrogen for 24 h. The mixture was filtered hot on asintered-glass frit and upon cooling, a yellow solid precipitated. Theresulting solid was triturated with acetonitrile/water (4:1, v/v),filtered on a sintered-glass frit and vacuum dried to yield 11 as abright yellow solid (0.632 g, 51.6% yield): ¹H NMR (300.13 MHz, CD₃OD)δ2.58 (s, 3H), 4.58 (s, 2H), 5.22 (s, 2H), 5.61 (s, 2H), 7.62 (m, 6H),7.80 (m, 2H), 8.18 (m, 2H), 8.58 (m, 2H); ¹³C-{¹h} NMR (75.4 MHz, CD₃OD)δ136.9, 136.2, 135.9, 132.9, 132.7, 131.4, 129.9, 128.3, 127.1, 126.8,126.5, 124.9, 124.1, 63.8, 57.1, 50.9, 40.7.

Example 2

This example provides the preparation of polymers used for theimmobilization of the amplification components.

2.1 Biocompatible Polymers (Silicone-Containing polymers and HydrogelCoatings)

2.1a Silicone-Containing Polymers

Synthesis of a Biocompatible Silicone/Polyurethane Patch Material forSubdermal Implantation

To an oven-dried, 100 mL, 3-neck round bottom flask fitted with amechanical stirrer, condenser, and under nitrogen was added 65 mL ofanhydrous THF, 80 mg of dibutyltin dilaurate (catalytic), 5.05 grams ofpoly(propylene glycol-b-ethylene glycol-b-propylene glycol)bis(2-aminopropyl ether) (8.4 mmol, 0.75 equiv.), 7.01 gramspolydimethylsiloxane, aminopropyldimethyl-terminated (average MW 2500)(2.8 mmol, 0.25 equiv.), and 2.94 grams of4,4′-methylenebis(cyclohexylisocyanate) (11.2 mmol, 1 equiv.) dried over4Å molecular sieves. An initial exotherm raised the temperature from 26to 39° C. The reaction solution was heated at reflux for about 15 hours,the heat was removed, and the solution was allowed to cool to roomtemperature. The cooled solution, now visibly more viscous, was pouredinto approximately 900 mL of rapidly stirring DI water. The precipitatedpolymer was collected and washed again in approximately 800 mL DI water.The collected polymer was dried in vacuo at 80° C.

Other suitable silicone-containing polymers are described in co-pendingapplication Ser. No. 08/721,262, now U.S. Pat. No. 5,777,060.

A bulk polymerization method of polymer formation was carried out withisophorone diisocyanate, PEG 600, diethylene glycol and aminopropylterminated polydimethyl siloxane as follows.

Isophorone diisocyanate (4.44 g, 20 mmol, 100 mol %) was dried overmolecular sieves and transferred to a 100 mL round bottom flask fittedwith a nitrogen purge line and a reflux condenser. PEG 600 (2.40 g, 4.0mmol, 20 mol %), diethylene glycol (1.06 g, 10 mmol, 50 mol %) andaminopropyl terminated polydimethylsiloxane (15 g, 6.0 mmol, 30 mol %,based on a 2500 average molecular weight) were added to the flask.Heating was initiated using a heating mantle until a temperature of 50°C. was obtained. Dibutyltin bis(2-ethylhexanoate) (15 mg) was added andthe temperature increased to about 95° C. The solution was continuouslystirred at a temperature of 65° C. for a period of 4 hr during whichtime the mixture became increasingly viscous. The resulting polymer wasdissolved in 50 mL of hot THF and cooled. After cooling, the solutionwas poured into 5 L of stirring DI water. The precipitated polymer wastorn into small pieces and dried at 50° C. until a constant weight wasachieved.

A solution polymerization method using 1,6-hexamethylene diisocyanate,PEG 200 and aminopropyl terminated polydimethylsiloxane was carried outas follows.

Dried 1,6-hexamethylene diisocyanate (1.34 g, 8 mmol, 100 mol %) wasadded to a 100 mL 3-neck flask containing 20 mL of dry THF. PEG 200 (0.8g, 4.0 mmol, 50 mol %) was added with stirring followed by addition ofaminopropyl terminated polydimethylsiloxane (10 g, 4.0 mmol, 50 mol %).The resulting solution was warmed to 50° C. and dibutyltinbis(2-ethylhexanoate) (about 15 mg) was added. After an initialtemperature rise to 83° C., the mixture was warmed and held at 70° C.for 12 hr, during which time the mixture had become very viscous. Aftercooling, the mixture was poured into 3 L of rapidly stirring DI water.The precipitated polymer was collected, washed with DI water (3×), torninto small pieces and dried at 50° C. until a constant weight wasobtained.

Table 1 provides five formulations for representative polymers aresuitable in biocompatible matrices. The polymers were prepared bysolution polymerization.

TABLE 1 Representative Polymer Formulations Poly- Poly(alkyleneAliphatic mer Diisocyanate glycol) diol Siloxane 1 1,6-Hexamethylene PEG600 (20%) DEG Aminopropyl (60%) (20%) 2 Isophorone PEG 600 (20%) DEGAminopropyl (50%) (30%) 3 1,6-Hexamethylene PEG 600 (50%) NoneAminopropyl (50%) 4 1,6-Hexamethylene PEG 400 (40%) None Aminopropyl(60%) 5 1,6-Hexamethylene PEG 600 (60%) None Aminopropyl (40%)

2.1b Hydrogel Coatings and Polymers

Hydrogels suitable for use as biosensor coatings were prepared bycombining a diisocyanate with an equivalent molar amount of ahydrophilic diol or diamine or with a combination of diol or diamine andchain extender such that the molar amount of the combination wasequivalent to the diisocyanate. The polymerizations were carried out ina one-pot reaction using THF as solvent and a trace catalyst(tributyltin ethylhexanoate). The reactions were heated to reflux andheld at this temperature overnight (about 16 hours). The resultingpolymer solution was poured into a large volume of DI water at about 20°C. and then filtered, dried, and washed with boiling DI water. Theresulting polymer was again dried then taken up in 2-propanol (as a 5 wt% solution) and used for encasing an amplification component.

Formulations of representative hydrogel coatings and polymers areprovided in Table 2.

TABLE 2 Representative Polymer Formulations Poly- Hydrophilic diol ormer Diisocyanate diamine Chain Extender 1 1,6-Hexamethylene Jeffamine600 (95%) Butanediol (5%) 2 1,6-Hexamethylene Jeffamine 2000 None (100%)3 1,6-Hexamethylene Jeffamine 2000 (90%) Butanediol (10%) 41,6-Hexamethylene PEG 2000 Butanediol (90%) (10%) 5 1,6-HexamethyleneJeffamine 230 Ethylene diamine (30%) (70%) 6 1,6-Hexamethylene PEG 600Ethylene diamine (75%) (25%) 7 Isophorone PEG 600 Butanediol (75%) (25%)8 Isophorone Jeffamine 900 1,6-Diaminohexane (70%) (25%) 9 IsophoroneJeffamine 900 1,2-Diaminocyclo- (50%) hexane (50%) 10 IsophoroneJeffamine 900 (50%) Isophorone diamine (50%)

2.2 Incorporation of Amplification Components into a BiocompatibleMatrix

2.2a Incorporation of FABA (fluorescein labeled boronic acid) into aMembrane.

The fluorescence of FABA is pH sensitive. In order to measure thefluorescence quenching due to the addition of glucose, a method wasdeveloped to incorporate the FABA in a polymeric membrane at pH 10. A 7%by weight solution of a hydrophilic polyurethane was made in 2-propanol.This base solution was combined with a 0.2 mmol solution of FABAdissolved in pH 10.0 phosphate buffer 0.1M). The final concentration ofthe membrane was approximately 5% by weight. A membrane was cast byspreading 3 ml of the solution onto a glass plate and allowing themembrane to dry. A portion of the membrane was then attached to a thinpiece of glass and placed in the diagonal of a fluorescence cuvette.Fluorescence spectra were run in pH 7.4 buffer solution. FIG. 15 showsthe calibration curve generated from this experiment. As seen, thefluorescence intensity is quenched by the glucose at pH 7.4.

Example 3

This example provides the description of covalent attachment of certaincomponents to biocompatible polymers.

Incorporation of (6) Into a Hydrophilic Polymer via a Urethane Linkage

To a three-necked 200 mL flask equipped with a condenser and a teflonstir bar was added 60 mL of dry THF, poly(propyleneglycol)-block-poly(ethylene glycol)-block-poly(propyleneglycol)bis(2-aminopropyl ether) (ave. Mn ca. 900, Jeffamine 900®) (6.30g, 7.0 mmol), and dibutyltin bis(2-ethylhexanoate) catalyst (0.052 g).While stirring, 2.49 g (9.5 mmol) of4,4′-methylenebis(cyclohexylisocyanate) was added and the resultingmixture was allowed to stir at room temperature overnight.9-((5-hydroxy-pentyl)aminomethyl)anthracene (0.32 g, 1.0 mmol) was addedand the mixture was refluxed for 2 h. The flask was removed from theheat, and the stir bar was replaced with a mechanical stirrer.1,6-hexamethylenediamine (0.29 g, 2.5 mmol) in 2 mL THF was added to thesolution with stirring and then refluxed for 1.5 h. The viscous mass wasadded to 500 mL water to produce an amber colored solid which was airdried on a Buchner funnel and placed in a vacuum oven overnight. Filmsof the polymer were prepared by casting ethanol solutions (1 gpolymer/10 mL ethanol) on glass plates and air drying.

Incorporation of (7) Into a Hydrophilic Polymer via a Urethane Linkage

To a three-necked 200 mL flask equipped with a condenser and a teflonstir bar was added 60 mL of dry THF, poly(propyleneglycol)-block-poly(ethylene glycol)-block-poly(propyleneglycol)bis(2-aminopropyl ether) (ave. Mn ca. 900, Jeffamine 900®) (6.30g, 7.0 mmol), and dibutyltin bis(2-ethylhexanoate) catalyst (0.052 g).While stirring, 2.49 g (9.5 mmol) of4,4′-methylenebis(cyclohexylisocyanate) was added and the resultingmixture was allowed to stir at room temperature overnight.9-((5-hydroxy-pentyl)-N-(o-boronobenzyl)amino)methyl)anthracene (0.44 g,1.0 mmol) was added and the mixture was refluxed for 2 h. The flask wasremoved from the heat, and the stir bar was replaced with a mechanicalstirrer. 1,6-hexamethylenediamine (0.29 g, 2.5 mmol) in 2 mL THF wasadded to the solution with stirring and then refluxed for 1.5 h. Theviscous mass was added to 500 mL water to produce an amber colored solidwhich was air dried on a Buchner funnel and placed in a vacuum ovenovernight. Films of the polymer were prepared by casting ethanolsolutions (1 g polymer/10 mL ethanol) on glass plates and air drying.

Incorporation of (11) Into a Hydrophilic Polymer via a Urethane Linkage

To a three-necked 200 mL flask equipped with a condenser and a teflonstir bar was added 60 mL of dry THF, poly(propyleneglycol)-block-poly(ethylene glycol)-block-poly(propyleneglycol)bis(2-aminopropyl ether) (ave. Mn ca. 900, Jeffamine 900®) (0.63g, 0.7 mmol), and dibutyltin bis(2-ethylhexanoate) catalyst (0.0052 g).While stirring, 0.249 g (0.95 mmol) of4,4′-methylenebis(cyclohexylisocyanate) was added and the resultingmixture was allowed to stir at room temperature overnight.10-(hydroxymethyl)-9-N-(o-boronobenzyl)amino)methyl)anthracene (0.04 g,0.1 mmol) was added and the mixture was refluxed for 2 h. The flask wasremoved from the heat, and the stir bar was replaced with a mechanicalstirrer. 1,6-hexamethylenediamine (0.029 g, 0.25 mmol) in 2 mL THF wasadded to the solution with stirring and then refluxed for 1.5 h. Theviscous mass was added to 500 mL water to produce an amber colored solidwhich was air dried on a Buchner funnel and placed in a vacuum ovenovernight. Films of the polymer were prepared by casting ethanolsolutions (1 g polymer/10 mL ethanol) on glass plates and air drying.

The above description is illustrative and not restrictive. Manyvariations of the invention will become apparent to those of skill inthe art upon review of this disclosure. Merely by way of example avariety of solvents, membrane formation methods, and other materials maybe used without departing from the scope of the invention. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

What is claimed is:
 1. A method of immobilizing a boronic acid moiety ina solid support, the method comprising: covalently attaching the boronicacid moiety to a solid support comprising a polymer matrix, wherein theboronic acid moiety comprises a compound of the formula:

 wherein D¹ is a dye selected from the group consisting of fluorescentdyes, luminescent dyes and colorimetric dyes; R¹, R³ and R⁴ are eachindependently substituents which can alter the electronic properties ofthe groups to which they are attached or are functional groups which canform covalent linkages to the polymer matrix; each R² is a memberindependently selected from the group consisting of hydrogen and C₁-C₄alkyl; each L¹ and L² is a linking group having from zero to fourcontiguous atoms selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus; Z is a heteroatom selected from thegroup consisting of nitrogen, sulfur, oxygen and phosphorus; x is aninteger from zero to four; and wherein the boronic acid moiety isattached to the polymer matrix by a covalent linkage to R¹.
 2. Themethod according to claim 1, wherein said solid support is abiocompatible membrane.
 3. The method according to claim 1, wherein saidsolid support is implantable within the body.
 4. The method of claim 1,wherein the boronic acid moiety is attached to the polymer matrix usinga covalent bonding technique selected from the group consisting ofcondensation, condensation-elimination and free radical polymerization.5. A method of immobilizing a boronic acid moiety in a solid support,the method comprising: covalently attaching the boronic acid moiety to asolid support comprising a polymer matrix, wherein the boronic acidmoiety comprises a compound of the formula:

 wherein D¹ is a dye selected from the group consisting of fluorescentdyes, luminescent dyes and colorimetric dyes; R¹, R³ and R⁴ are eachindependently substituents which can alter the electronic properties ofthe groups to which they are attached or are functional groups which canform covalent linkages to the polymer matrix; each R² is a memberindependently selected from the group consisting of hydrogen and C₁-C₄alkyl; each L¹ and L² is a linking group having from zero to fourcontiguous atoms selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus; Z is a heteroatom selected from thegroup consisting of nitrogen, sulfur, oxygen and phosphorus; x is aninteger from zero to four; and wherein the boronic acid moiety isattached to the polymer matrix by a covalent linkage to R³.
 6. Themethod of claim 5, wherein the boronic acid moiety is attached to thepolymer matrix using a covalent bonding technique selected from thegroup consisting of condensation, condensation-elimination and freeradical polymerization.
 7. The method according to claim 5, wherein saidsolid support is a biocompatible membrane.
 8. The method according toclaim 5, wherein said solid support is implantable within the body.
 9. Amethod of immobilizing a boronic acid moiety in a solid support, themethod comprising: covalently attaching the boronic acid moiety to asolid support comprising a polymer matrix, wherein the boronic acidmoiety comprises a compound of the formula:

 wherein D¹ is a dye selected from the group consisting of fluorescentdyes, luminescent dyes and colorimetric dyes; R¹, R³ and R⁴ are eachindependently substituents which can alter the electronic properties ofthe groups to which they are attached or are functional groups which canform covalent linkages to the polymer matrix; each R² is a memberindependently selected from the group consisting of hydrogen and C₁-C₄alkyl; each L¹ and L² is a linking group having from zero to fourcontiguous atoms selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus; Z is a heteroatom selected from thegroup consisting of nitrogen, sulfur, oxygen and phosphorus; x is aninteger from zero to four; and wherein the boronic acid moiety isattached to the polymer matrix by a covalent linkage to R⁴.
 10. Themethod of claim 9, wherein the boronic acid moiety is attached to thepolymer matrix using a covalent bonding technique selected from thegroup consisting of condensation, condensation-elimination and freeradical polymerization.
 11. The method according to claim 9, whereinsaid solid support is a biocompatible membrane.
 12. The method accordingto claim 9, wherein said solid support is implantable within the body.13. A method of immobilizing a boronic acid moiety in a solid support,the method comprising: entrapping the boronic acid moiety in a solidsupport comprising a polymer matrix, wherein the boronic acid moietycomprises a compound of the formula:

 wherein D¹ is a dye selected from the group consisting of fluorescentdyes, luminescent dyes and colorimetric dyes; R¹, R³ and R⁴ are eachindependently substituents which can alter the electronic properties ofthe groups to which they are attached or are functional groups which canform covalent linkages to the polymer matrix; each R² is a memberindependently selected from the group consisting of hydrogen and C₁-C₄alkyl; each L¹ and L² is a linking group having from zero to fourcontiguous atoms selected from the group consisting of carbon, oxygen,nitrogen, sulfur and phosphorus; Z is a heteroatom selected from thegroup consisting of nitrogen, sulfur, oxygen and phosphorus; and x is aninteger from zero to four.